Contrast enhanced trans rectal ultrasound (TRUS), multimodality 3T magnetic resonance imaging, magnetic resonance spectroscopy and nuclear bone scans are current imaging modalities used in contemporary urological practice for the diagnosis and staging of prostate cancer. Such imaging modalities may be considered prostate imaging modalities, but currently lack the prostate cancer specific imaging modalities. With an increasing number of patients with minimal prostate cancer and opting for either focal treatment or active surveillance, the need for accurate, cancer specific imaging tools for diagnosis, treatment monitoring and follow-up is needed.
Prostate specific antigen (PSA) is a 33,000 kDa single chain glycoprotein first characterized from human prostate tissue. PSA is synthesized and secreted as a unique differentiation product of the prostatic glandular cells, both from normal and cancerous cells. Low levels of PSA are detected in normal and cancerous breast tissue also.
Prostate Specific Antigen (PSA) is a chymotrypsin-like serine protease that is measurable in the blood and is used as a clinical test to detect prostate cancer and follow response to therapy. However, PSA is not active in the blood and is only active within tumor sites and in the normal prostate tissue. The concept of capitalizing upon the prostate specific expression of the protease PSA to target therapeutic agents to prostate cancer sites was first proposed in 1992. Since that time, considerable development, research and systematic effort have been applied to bring that idea to fruition. These efforts have resulted in identification of initial PSA-activated pro-drugs which have been described in detail elsewhere (see, for example, U.S. Pat. No. 6,410,514).
Human Glandular Kallikrein 2 (hK2) is the protein product of the human kallikrein gene hKLK2, one of three related kallikrein genes that also include hKLK1 and hKLK3. These three genes are clustered on chromosome 19q13.2 q13.4. The protein product of hKLK3 is prostate-specific antigen (PSA). While PSA is the predominant tissue kallikrein in the prostate, hK2 is also found almost exclusively in the prostate. hK2 is a glycoprotein containing 237 amino acids and a mass of 28.5 kpa. hK2 and PSA share some properties, such as high amino acid sequence identity, prostate localization, androgen regulation and gene expression, but are quite distinct from one another biochemically.
hK2 and PSA differ most markedly in their enzyme properties. Unlike PSA, a chymotrypsin-like protease, hK2 displays the trypsin-like specificity common to most members of the kallikrein family of proteases. hK2 can cleave semenogelin proteins, with an activity that is comparable to PSA. The level of hK2 in the seminal fluid is only 1% of the level of PSA. hK2 has trypsin-like activity, similar to hK1, although it does not appear to function as a classic kininogenase.
In the normal prostate, the levels of expressed hK2 protein are lower than those of PSA. However, hK2 is more highly expressed by prostate cancer cells than by normal prostate epithelium. Comparison of immunohistochemical staining patterns demonstrated incrementally increased staining in poorly differentiated prostate cancers. The intensity of staining has been found to increase with increasing Gleason score, in contrast to PSA, which tends to show decreased staining with increasing Gleason grade, suggesting that hK2 might potentially be a better tumor marker for prostate cancer than PSA.
Recently, three independent groups reported that recombinant hK2 could convert inactive pro-PSA in to the mature PSA protease by release of the propeptide in vitro, thus establishing a possible physiologic connection between hK2 and PSA. hK2 is also secreted in an inactive precursor form. Pro-hK2 may have autocatalytic activity, but the mechanism of activation in vivo is unknown and may involve several additional enzymes. hK2 has also been shown to activate single chain urokinase-type plasminogen activator, scuPA, to the active two-chain form, uPA, which is highly correlated with prostate cancer metastasis. More recently, hK2 has been shown to inactivate the major tissue inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1). Thus hK2 may influence the progression of prostate cancer by the activation of uPA and by the inactivation of PAI-1.
Prostate Specific Membrane Antigen (PSMA) is a 100 kDa prostate epithelial cell type II transmembrane glycoprotein that was originally isolated from a cDNA library from the androgen responsive LNCaP human prostate cancer cell line (Tombal et al., Prostate 43:303-317, 2000). Immunohistochemical studies using monoclonal antibodies have demonstrated that PSMA is expressed by normal prostate epithelium and is even more highly expressed by a large proportion of prostate cancers, including metastatic prostate cancers (Tombal et al., Prostate, 43:303-317, 2000; Wright et al., Urol. Oncol., 1:18-28, 1995; Lopes et al., Cancer Res., 50:6423-6429, 1990). Low-level detection of the PSMA protein has also been seen in the duodenal mucosa and in a subset of proximal renal tubules (Silver et al., Clin. Cancer Res., 3:81-85, 1997; Chang et al., Cancer Res., 59:3192-3198, 1999). PSMA enzymatic activity is also present in the brain. In all other human tissues, including normal vascular endothelium, PSMA expression was not detectable. In two separate studies using different monoclonal antibodies, PSMA expression was also undetectable in other non-prostatic primary tumors (Silver et al., Clin. Cancer Res., 3:81-85, 1997; Chang et al., Cancer Res., 59:3192-3198, 1999). In a number of studies, however, PSMA expression, has been detected in the neovasculature of a large number of different tumor types including breast, renal, colon and transitional cell carcinomas (Silver et al., Clin. Cancer Res., 3:81-85, 1997; Chang et al., Cancer Res., 59:3192-3198, 1999). A final interesting aspect of PSMA expression is that the PSMA mRNA is upregulated upon androgen withdrawal (Israeli et al., Cancer Res., 54:1807-1811, 1994; Cunha et al., Cancer Lett. 236:229-38, 2006). In contrast, PSA expression is downregulated by androgen deprivation (Chang et al., Clin. Cancer Res., 5:2674-2681, 1999; Godeiro et al., J. Carcinog., 5:21-24, 2006). Therefore, PSMA should be readily targetable in the majority of hormone refractory patients because PSMA levels are expected to remain high following androgen ablation.
Two discrete enzymatic functions for PSMA have been described. Initially, Carter et al., Proc. Natl. Acad. Sci., USA, 93:749-753 (1996), demonstrated that PSMA possesses the hydrolytic properties of an N-acetylated α-linked acidic dipeptidase (NAALADase). NAALADase is a membrane hydrolase activity that is able to hydrolyze the neuropeptide N-acetyl-1-aspartyl-1-glutamate (NAAG) to yield the neurotransmitter glutamate and N-acetyl-aspartate (Robinson et al., J. Biol. Chem., 262:14498-14506, 1987; Pinto et al., Clin. Cancer Res., 2:1445-1451, 1996). In addition to the NAALADase activity, PSMA also functions as a pteroyl poly-γ-glutamyl carboxypeptidase (folate hydrolase) (Luthi-Carter et al., Brain Res., 795:341-348, 1998.). PSMA exhibits exopeptidase activity and has been classified as a glutamate carboxypeptidase II (Heston et al., Urology 49 (Suppl 3A):104-112, 1997). It is able to progressively hydrolyze γ-glutamyl linkages of both poly-γ-glutamated folates and methotrexate analogs with varying length glutamate chains (Luthi-Carter et al., Brain Res., 795:341-348, 1998, Mhaka et al., Cancer Biol. Ther., 3:551-8, 2004).
The observation that the PSMA protein continually internalizes, even in the absence of bound antibody, indicates that labeled small molecule inhibitors of PSMA's activity may be used to image prostate cancer. Recently it was demonstrated that both 11C and 125I radiolabeled urea derivatives with high affinity for PSMA can detect PSMA producing xenografts in nude mice with tumor/muscle ratios of 10.8 and 4.7 respectively at 30 minutes post injection (Singh et al., J. Med. Chem., 48:3005-14, 2005). These agents were also readily taken up by the mouse kidney, which is known to produce the highest levels of PSMA in the mouse. The kidney uptake appeared to be due to inhibitor binding to PSMA as this binding could be blocked by coadministration of high dose of a second, unlabeled, potent PSMA inhibitor (i.e., PMPA) (Singh et al., J. Med. Chem., 48:3005-14, 2005).
These inhibitory compounds, like antibodies, bind to PSMA with 1:1 stoichiometry. As an alternative approach to targeting, the unique enzymatic activity of PSMA can be exploited for signal amplification through the delivery of imaging and/or cytotoxic agents (e.g., prodrugs) that require PSMA for activation selectively within tumor sites.
Thapsigargin (TG) is a sesquiterpene-γ-lactone available by extraction from the seeds and roots of the umbelliferous plant Thapsia garganica L. Thapsigargin selectively inhibits the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) pump, found in skeletal, cardiac, muscle and brain microsomes. The apparent dissociation constant is 2.2 pM or less.
TG operates by what is believed to be a unique method of killing cells. TG induced inhibition of the SERCA pump leads to depletion of the ER Ca2+ pool. This depletion apparently results in the generation of a signal, possibly from an ER-derived diffusible messenger, so that the plasma membrane is more permeable to extracellular divalent cations. The resulting influx of these cations is responsible for the death of cells.